Vascular network formation is a limiting obstacle for tissue engineering strategies targeting repair and regeneration of damaged or diseased tissue. Development of functional vascular networks is important for the treatment of various diseases, such as, diabetic ulcers, limb ischemia, cerebral ischemia, peripheral vascular disease, and cardiovascular disease. Therapeutic use of stem and progenitor cells for the treatment of diseases or dysfunctional tissues has been limited by the ability to control their survival, proliferation, and differentiation. Recently, three-dimensional (3D) extracellular matrices (ECMs) have been identified as an important component of stem cell technology to assist in guiding cell behavior. However, tissue engineering approaches with engineered collagen matrices to generate functional vascular networks, needed for the treatment of peripheral and cardiovascular disease, have not been previously developed.
Applicants have engineered collagen-based matrices with the potential to direct vessel formation. Mechanical properties including fiber diameter, fibril density, fibril length, and matrix stiffness can be modulated by controlling polymerization parameters including collagen concentration, temperature, pH, ionic strength, and polymerization time. Applicants describe engineered collagen-based matrices that modulate in vitro and in vivo vessel formation to improve the efficiency of cellular-based therapies to regenerate or repair blood vessels. Systemic variation of polymerization conditions such as pH, ionic strength, and molecular composition provides a means to control polymerization kinetics, fibril microstructure, and mechanical properties of 3D collagen matrices. These microstructural-mechanical properties, in turn, provide instructional information to stem cells, and have been used by Applicants as design parameters to influence cell behavior.
In one illustrative embodiment, a composition for supporting stem cells is provided, the composition comprising an engineered, purified collagen-based matrix comprising collagen fibrils, and a population of stem cells, wherein the fibril volume fraction of the matrix is about 1% to about 60%, and wherein the storage modulus of the matrix is about 10 Pa to about 700 Pa.
In the above described embodiment, the stem cells can be mesenchymal stem cells, the fibril volume fraction of the matrix can be about 20%, the storage modulus of the matrix can be about 40 Pa to about 50 Pa, the stem cells can be differentiated into adipocytes, the fibril volume fraction of the matrix can be about 50% to about 60%, the storage modulus of the matrix can be about 650 Pa to about 700 Pa, the stem cells can be differentiated into osteoblasts, or the seeding density of the stem cells can be about 0.3×104 cells/ml to about 60×104 cells/ml.
In another illustrative embodiment, a composition for supporting stem cells is provided, the composition comprising an engineered, purified collagen-based matrix comprising collagen fibrils, and a population of stem cells, wherein the fibril volume fraction of the matrix is about 1% to about 60%, and wherein the loss modulus of the matrix is about 1 Pa to about 75 Pa.
In the above described embodiment, the stem cells can be mesenchymal stem cells, the fibril volume fraction of the matrix can be about 20%, the storage modulus of the matrix can be about 40 Pa to about 50 Pa, the stem cells can be differentiated into adipocytes, the fibril volume fraction of the matrix can be about 50% to about 60%, the storage modulus of the matrix can be about 650 Pa to about 700 Pa, the stem cells can be differentiated into osteoblasts, or the seeding density of the stem cells can be about 0.3×104 cells/ml to about 60×104 cells/ml.
In a further illustrative embodiment, a composition for supporting stem cells is provided, the composition comprising an engineered, purified collagen-based matrix comprising collagen fibrils, and a population of stem cells, wherein the fibril volume fraction of the matrix is about 1% to about 60%, and wherein the compressive modulus of the matrix is about 2500 Pa to about 18,000 Pa.
In the above described embodiment, the stem cells can be mesenchymal stem cells, the fibril volume fraction of the matrix can be about 20%, the storage modulus of the matrix can be about 40 Pa to about 50 Pa, the stem cells can be differentiated into adipocytes, the fibril volume fraction of the matrix can be about 50% to about 60%, the storage modulus of the matrix can be about 650 Pa to about 700 Pa, the stem cells can be differentiated into osteoblasts, or the seeding density of the stem cells can be about 0.3×104 cells/ml to about 60×104 cells/ml.
In yet another illustrative embodiment, a tissue graft composition is provided, the composition comprising an engineered, purified collagen-based matrix comprising collagen fibrils, and a population of stem cells, wherein the fibril volume fraction of the matrix is about 1% to about 60%, and wherein the storage modulus of said matrix is about 10 Pa to about 700 Pa.
In the above described embodiment, the stem cells can be mesenchymal stem cells, the fibril volume fraction of the matrix can be about 20%, the storage modulus of the matrix can be about 40 Pa to about 50 Pa, the stem cells can be differentiated into adipocytes, the fibril volume fraction of the matrix can be about 50% to about 60%, the storage modulus of the matrix can be about 650 Pa to about 700 Pa, the stem cells can be differentiated into osteoblasts, or the seeding density of the stem cells can be about 0.3×104 cells/ml to about 60×104 cells/ml.
In a further illustrative embodiment, a tissue graft composition is provided, the composition comprising an engineered, purified collagen-based matrix comprising collagen fibrils, and a population of stem cells, wherein the fibril volume fraction of the matrix is about 1% to about 60%, and wherein the loss modulus of the matrix is about 1 Pa to about 75 Pa.
In the above described embodiment, the stem cells can be mesenchymal stem cells, the fibril volume fraction of the matrix can be about 20%, the storage modulus of the matrix can be about 40 Pa to about 50 Pa, the stem cells can be differentiated into adipocytes, the fibril volume fraction of the matrix can be about 50% to about 60%, the storage modulus of the matrix can be about 650 Pa to about 700 Pa, the stem cells can be differentiated into osteoblasts, or the seeding density of the stem cells can be about 0.3×104 cells/ml to about 60×104 cells/ml.
In a further illustrative embodiment, a tissue graft composition is provided, the composition comprising an engineered, purified collagen-based matrix comprising collagen fibrils, and a population of stem cells, wherein the fibril volume fraction of the matrix is about 1% to about 60%, and wherein the compressive modulus of the matrix is about 2500 Pa to about 18,000 Pa.
In the above described embodiment, the stem cells can be mesenchymal stem cells, the fibril volume fraction of the matrix can be about 20%, the storage modulus of the matrix can be about 40 Pa to about 50 Pa, the stem cells can be differentiated into adipocytes, the fibril volume fraction of the matrix can be about 50% to about 60%, the storage modulus of the matrix can be about 650 Pa to about 700 Pa, the stem cells can be differentiated into osteoblasts, or the seeding density of the stem cells can be about 0.3×104 cells/ml to about 60×104 cells/ml.
In another illustrative embodiment, a method of preparing a tissue graft composition is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, and contacting the matrix with a population of stem cells, wherein the fibril volume fraction of the matrix is about 1% to about 60%, and wherein the storage modulus of the matrix is about 10 Pa to about 700 Pa.
In the above described embodiment, the stem cells can be mesenchymal stem cells, the fibril volume fraction of the matrix can be about 20%, the storage modulus of the matrix can be about 40 Pa to about 50 Pa, the stem cells can be differentiated into adipocytes, the fibril volume fraction of the matrix can be about 50% to about 60%, the storage modulus of the matrix can be about 650 Pa to about 700 Pa, the stem cells can be differentiated into osteoblasts, or the seeding density of the stem cells can be about 0.3×104 cells/ml to about 60×104 cells/ml.
In another illustrative embodiment, a method of preparing a tissue graft composition is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, and contacting the matrix with a population of stem cells, wherein the fibril volume fraction of the matrix is about 1% to about 60%, and wherein the loss modulus of the matrix is about 1 Pa to about 75 Pa.
In the above described embodiment, the stem cells can be mesenchymal stem cells, the fibril volume fraction of the matrix can be about 20%, the storage modulus of the matrix can be about 40 Pa to about 50 Pa, the stem cells can be differentiated into adipocytes, the fibril volume fraction of the matrix can be about 50% to about 60%, the storage modulus of the matrix can be about 650 Pa to about 700 Pa, the stem cells can be differentiated into osteoblasts, or the seeding density of the stem cells can be about 0.3×104 cells/ml to about 60×104 cells/ml.
In another illustrative embodiment, a method of preparing a tissue graft composition is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, and contacting the matrix with a population of stem cells, wherein the fibril volume fraction of the matrix is about 1% to about 60%, and wherein the compressive modulus of the matrix is about 2500 Pa to about 18,000 Pa.
In the above described embodiment, the stem cells can be mesenchymal stem cells, the fibril volume fraction of the matrix can be about 20%, the storage modulus of the matrix can be about 40 Pa to about 50 Pa, the stem cells can be differentiated into adipocytes, the fibril volume fraction of the matrix can be about 50% to about 60%, the storage modulus of the matrix can be about 650 Pa to about 700 Pa, the stem cells can be differentiated into osteoblasts, or the seeding density of the stem cells can be about 0.3×104 cells/ml to about 60×104 cells/ml.
In another illustrative embodiment, a tissue graft composition is provided, the composition comprising an engineered, purified collagen-based matrix comprising collagen fibrils, and one or more vessels.
In the above described embodiment, the fibril volume fraction of the matrix can be about 1% to about 60% and the storage modulus of the matrix can be about 10 Pa to about 700 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the loss modulus of the matrix can be about 1 Pa to about 75 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the compressive modulus of the matrix can be about 2500 Pa to about 18,000 Pa, or the composition can further comprises endothelial progenitor cells.
In another illustrative embodiment, a method of preparing a tissue graft composition is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, and contacting the matrix with endothelial progenitor cells wherein one or more vessels are formed within the matrix.
In the above described embodiment, the fibril volume fraction of the matrix can be about 1% to about 60% and the storage modulus of the matrix can be about 10 Pa to about 700 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the loss modulus of the matrix can be about 1 Pa to about 75 Pa, or the fibril volume fraction of the matrix can be about 1% to about 60% and the compressive modulus of the matrix can be about 2500 Pa to about 18,000 Pa.
In another illustrative embodiment, a method of promoting vessel formation within a tissue graft composition is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, and contacting the matrix with endothelial progenitor cells wherein one or more vessels are formed within the matrix.
In the above described embodiment, the fibril volume fraction of the matrix can be about 1% to about 60% and the storage modulus of the matrix can be about 10 Pa to about 700 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the loss modulus of the matrix can be about 1 Pa to about 75 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the compressive modulus of the matrix can be about 2500 Pa to about 18,000 Pa, or the vessels can be formed from endothelial progenitor cells.
In another illustrative embodiment, a method of vascularizing a tissue graft composition prior to implantation is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, and contacting the matrix with endothelial progenitor cells wherein one or more vessels are formed within the matrix.
In the above described embodiment, the fibril volume fraction of the matrix can be about 1% to about 60% and the storage modulus of the matrix can be about 10 Pa to about 700 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the loss modulus of the matrix can be about 1 Pa to about 75 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the compressive modulus of the matrix can be about 2500 Pa to about 18,000 Pa, or the vessels can be formed from endothelial progenitor cells.
In another illustrative embodiment, a method of producing a population of stem cells is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, and contacting the matrix with endothelial progenitor cells wherein the population of cells is produced.
In the above described embodiment, the method can further comprise the step of isolating the stem cells from the matrix, the stem cells can be isolated from the matrix using a collagenase solution, the fibril volume fraction of the matrix can be about 1% to about 60% and the storage modulus of the matrix can be about 10 Pa to about 700 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the loss modulus of the matrix can be about 1 Pa to about 75 Pa, or the fibril volume fraction of the matrix can be about 1% to about 60% and the compressive modulus of the matrix can be about 2500 Pa to about 18,000 Pa.
In a further illustrative embodiment, a method of enhancing CD34 expression on stem cells is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, and contacting the matrix with endothelial progenitor cells wherein the cells exhibit enhanced CD34 expression.
In the above described embodiment, the method can further comprise the step of isolating the stem cells from the matrix, the stem cells can be isolated from the matrix using a collagenase solution, the fibril volume fraction of the matrix can be about 1% to about 60% and the storage modulus of the matrix can be about 10 Pa to about 700 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the loss modulus of the matrix can be about 1 Pa to about 75 Pa, or the fibril volume fraction of the matrix can be about 1% to about 60% and the compressive modulus of the matrix can be about 2500 Pa to about 18,000 Pa.
In a further illustrative embodiment, a method of producing blood vessels de novo is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, contacting the matrix with endothelial progenitor cells wherein the vessels are formed, and isolating the vessels from the matrix.
In the above described embodiment, the vessels can be isolated from the matrix using a collagenase solution, the fibril volume fraction of the matrix can be about 1% to about 60% and the storage modulus of the matrix can be about 10 Pa to about 700 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the loss modulus of the matrix can be about 1 Pa to about 75 Pa, or the fibril volume fraction of the matrix can be about 1% to about 60% and the compressive modulus of the matrix can be about 2500 Pa to about 18,000 Pa.
In a further illustrative embodiment, a method of treating a tissue of a patient wherein the tissue is in need of vascularization is provided, the method comprising the steps of engineering a purified collagen-based matrix comprising collagen fibrils, contacting the matrix with endothelial progenitor cells wherein vessels are formed de novo, isolating the vessels from the matrix, and implanting the vessels into the tissue of the patient.
In the above described embodiment, the vessels can be isolated from the matrix using a collagenase solution, the fibril volume fraction of the matrix can be about 1% to about 60% and the storage modulus of the matrix can be about 10 Pa to about 700 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the loss modulus of the matrix can be about 1 Pa to about 75 Pa, or the fibril volume fraction of the matrix can be about 1% to about 60% and the compressive modulus of the matrix can be about 2500 Pa to about 18,000 Pa.
In another embodiment, a method of forming vessels in vivo is provided. The method comprises the step of implanting an engineered, purified collagen-based matrix comprising collagen fibrils and endothelial progenitor cells into a patient wherein vessel formation at the implantation site is enhanced in vivo.
In the above described embodiment, the fibril volume fraction of the matrix can be about 1% to about 60% and the storage modulus of the matrix can be about 10 Pa to about 700 Pa, the fibril volume fraction of the matrix can be about 1% to about 60% and the loss modulus of the matrix can be about 1 Pa to about 75 Pa, or the fibril volume fraction of the matrix can be about 1% to about 60% and the compressive modulus of the matrix can be about 2500 Pa to about 18,000 Pa.